5.55.5 © Springer-Verlag Berlin Heidelberg 2005 II.5.5 Boric acid by Shinichi Suzuki Introduction Boric acid and borax ( sodium tetraborate) had been widely used as antiseptics (preservatives) and/or ethical drugs; however, by reevaluation made in Japan, 1985, the questionability was raised on their e ectiveness. Nowadays, they are being permitted to be used only for washing or disinfection of the eye as ethical drugs. In recent year, boric acid has been widely used as a household insecticide especially for cockroaches (called “ boric acid ball”), resulting in the in- crease of boric acid poisoning cases.  e boric acid ball is a toxic bait for cockroaches; its toxicity is slow-acting, but fatal for the insects. Various types of boric acid ball products are commercially available, but it can be hand- made at home very easily. As an attractant material for cockroaches, crushed onions are being usually added to boric acid. Because of wide availability of the boric acid balls, accidental intake of them by infants can occur frequently. As poisoning symptoms of boric acid, nausea, vomiting, diarrhea and bleed- ing can be mentioned. When a relatively large amount of boric acid is ingested by an infant, there is a possibility of fatality. In Japanese trials, only with the presence of a toxic substance in a vomitus, it is di cult to prove that the toxic substance is causative of a poisoning case; namely, it does not verify the intake of the poison by the victim.  erefore, in Japanese law, it is essential to verify the pres- ence of a poison in blood, urine and/or stomach contents (not vomitus). In this chapter, the methods for analysis of boric acid in blood, urine and stomach contents by spectrophotometry and HPLC are described. Spectrophotometric analysis [1, 2] Reagents and their preparation • Boric acid and borax can be obtained from Sigma (St. Louis, MO, USA). • Curcumin solution: 0.1 g curcumin (Sigma) is dissolved in puri ed water to prepare 100 mL solution. • 2-Ethyl-1,3-hexanediol solution: 10 mL of 2-ethyl-1,3-hexanediol (Aldrich, Milwaukee, WI, USA) is dissolved in chloroform to prepare 100 mL solution. Analytical instrument A DU-70 type UV-Vis spectrophotometer a (Beckman, Fullerton, CA, USA). 432 Boric acid Procedure  e procedure for blood, urine and stomach contents is described here, but it is, of course, applicable to the solubilized specimens of a boric acid ball itself. i. A part of specimens (blood, urine and stomach contents) is mixed well with puri ed water to serve as sample solution. ii. A 0.5-mL volume of the sample solution is diluted with puri ed water to prepare 5 mL solution. iii. To this solution, are added 1 mL of 50 % sulfuric acid and 5 mL 2-ethyl-1,3-hexanediol, followed by shaking for extraction b . iv.  e tube containing the above mixture is centrifuged at 3,000 rpm for 5 min; the resulting 2-ethyl-1,3-hexanediol layer is passed through a  lter paper. v. A 1-mL volume of the  ltrate is mixed with 1 mL of the curcumin solution and 0.5 mL of concentrated sulfuric acid, le for 30 min, then mixed with 25 mL ethanol and  nally le for 10 min. vi.  e absorbance of the above solution is measured with a spectrophotometer at 550 nm to detect and quantitate boric acid. Assessment of the method  is method is simple and excellent in quantitativeness; the detection limit is about 1 µg/mL as a concentration in the test tube. > Figure 5.1 shows absorption spectra for the complex form of boric acid, which had been contained in various matrices. Absorption spectra for boric acid in various matrices after its chelating extraction and curcumin color reaction. 1: Boric acid standard solution (30 µg/mL); 2: a blood specimen; 3: a stomach content specimen; 4: a urine specimen. ⊡ Figure 5.1 433 By the present spectrophotometric analysis of boric acid in a urine specimen, the value obtained may be slightly greater than the true value.  is is probably due to interference by a certain urinary element, which also forms a similar chelated complex. HPLC analysis Reagents and their preparation • Mobile phase: 5 mM perchloric acid solution is prepared, passed through a membrane  l- ter and degassed. • 2-Ethyl-1,3-hexanediol solution: the preparation is the same as described in the spectro- photometric analysis section. HPLC conditions Instrument: an LC-5A high-performance liquid chromatograph c (Shimadzu Corp., Kyoto, Japan); detector: a Waters 410 type di erential refractometer (Waters, Milford, MA, USA). Column: Shim-pack SCR-102H (30 cm × 8 mm i.d., Shimadzu Corp., a strong anion ex- changer). Mobile phase: 5 mM perchloric acid solution; its  ow rate: 0.5 mL/min; column (oven) temperature: 50 °C. Procedure i.  e steps i.–iv. described in the spectrophotometric analysis section are followed.  e boric acid chelate is extracted and  ltered. ii.  e 5 mL of the 2-ethyl-1,3-hexanediol  ltrate is evaporated to dryness. iii.  e residue is dissolved in an appropriate amount of the mobile phase; a 5-µL aliquot of the solution is injected into HPLC. iv. For quantitation, each of various concentrations of the authentic boric acid is added to the blank matrix, and then treated in the same way as described above to construct an external calibration curve. Assessment of the method > Figure 5.2 shows an HPLC chromatogram for the complex of the authentic boric acid (injected amount, 1 µg). > Figure 5.3 shows that obtained from a blood specimen; many impurity peaks appear for biomedical specimens, but speci c analysis can be achieved, if the analytical conditions are set appropriately. By this method, the detection limits are about 10 µg/mL (50 ng in an injected volume). HPLC analysis 434 Boric acid In this method, the analysis is being made with an HPLC instrument equipped with a dif- ferential refractometer a er chelation of boric acid. To detect borate ion without chelation, ion chromatography should be used; in the latter method, a conductivity detector is usually used. In the modern type of an ion chromatograph, an auto-suppressor mode is being adopted, resulting cleaner backgrounds and higher sensitivity (more than 10 times higher than that of the present HPLC). However, the cost for an ion chromatographic system is much higher than that of the conventional HPLC. Poisoning case, and toxic and fatal concentrations A 83-year-old male [3] ingested 3 balls of hand-made boric acid mixture around noon, and vomited several times a er supper in the evening. On the next day, he visited a hospital, be- cause exanthemas appeared in his face, chest and abdomen. At that time, general conditions seemed good, but the laboratory tests showed BUN at 34.2 mg/dL and Cr at 1.6 mg/dL, show- HPLC chromatogram for the standard boric acid after chelating extraction. The amount of boric acid in an injected volume was 1 µg. ⊡ Figure 5.2 HPLC chromatogram for a chelated extract of blood containing boric acid. ⊡ Figure 5.3 435 ing slight renal dysfunction. Five days a er admission, the erythematous exanthemas extended to all parts of his body, followed by remarkable desquamation. From the 6th day of admission, general fatigue, dyspnea and respiratory noises appeared. By X-ray irradiation of the chest, pleural e usion was discovered, suggesting the presence of cardiac dysfunction. In addition, he fell into hepatic dysfunction and DIC.  e anti-DIC treatments and hemodialysis (2 times) were performed for him. His bad conditions were improved gradually; he was discharged on day 42. As acute boric acid poisoning symptoms, nausea, vomiting, diarrhea and abdominal pain is most common as observed in the above case; headache, scarlet fever-like exanthemas and desquamation of the skin are sometimes observed. When the poisoning becomes severe, list- lessness, convulsions, shock and renal failure appear successively.  e fatal doses of boric acid are 15–20 g for adults, 5–6 g for small children and 2–3 g for babies. When a cockroach ball with a high boric acid content is eaten by a small child, there is a possibility of death. Such concern is not limited to the boric acid ball; caution should be directed also toward boric acid solutions, which are being widely used as preservatives and disinfectants. In survived cases, in which boric acid had been taken by mistake or for suicidal purpose, the boric acid concentrations were 1–44 µg/mL in blood and 20 µg/mL in urine [3]. In a fatal case, its blood concentration was 37.7 µg/mL 30 h a er ingestion [3]. Notes a) Any type of spectrophotometers can be used. b)  e chelating complex of 2-ethyl-1,3-hexanediol and boric acid is considered to be formed by the following reaction. c) Any type of HPLC instruments can be used. References 1) Public Health Council (ed) (1983) Methods of Analysis in Health Science, Methods for Food Additives, Preserva- tives, Boric Acid and Its Salts. The Pharmaceutical Society of Japan, Tokyo, pp 81–83 (in Japanese) 2) Fukui S (1983) A method for determination of boric acid in foods using chelating extraction with 2-ethyl-1,3- hexanediol and protonated curcumin. Eisei Kagaku 29:323–328 (in Japanese with an English abstract) 3) Japan Poison Information Center (ed) (2000) Poisoning Accidents and their Countermeasures with Special Reference to Actual Cases, revised edn. Jiho Inc., Tokyo, pp 349–352 (in Japanese) Poisoning case, and toxic and fatal concentrations . commercially available, but it can be hand- made at home very easily. As an attractant material for cockroaches, crushed onions are being usually added to. discharged on day 42. As acute boric acid poisoning symptoms, nausea, vomiting, diarrhea and abdominal pain is most common as observed in the above case;